Cls430641u

Human lymphatic endothelial cells (HLECs, ScienCell, 2500) were cultured in standard T75 cm² cell culture flasks (Corning, CLS430641U) coated with fibronectin as previously described [30 ,31 ] at a starting cell concentration of 5·10⁵ cells. Cultures were maintained in endothelial basal medium-2 (Lonza, CC-3156) with EGM-2 MV SingleQuot Kit (Lonza, CC-4147), hereafter referred to as endothelial media. HLECs were cultured to 90-95% confluency at passage 3 for all experiments. Primary fibroblasts were routinely cultured in DMEM (Gibco, 11965092) with 10% fetal bovine serum (FBS, VWR, 97068-085), 1% Pen/Strep (ThermoFisher, 15140-122), and 1 µg/mL hydrocortisone (STEMCELL Technologies Inc., 07925). Primary human normal oral fibroblasts (HOrF, ScienCell, 2640) were cultured in standard T75 cm² cell culture flasks (Corning, CLS430641U) at a starting cell concentration of 5·10⁵ cells in fibroblast media (ScienCell, 2301). All cultures were kept in a humidified incubator at 37°C with 5% CO₂. Media was refreshed every 2-3 days, and cells were cultured to 90-95% confluency.

HNSCC diagnosis was confirmed by a pathologist for all patients. Before processing, the residual samples were maintained in transport media (DMEM basal media with gentamycin, amphotericin, and penicillin/streptomycin (Pen/Strep) at 1% v/v each). Samples were then minced and were transferred to a 15 mL conical tube containing digestion media (6 mL of transport media with 0·1% collagenase (Thermo-Fisher, 17100017), 0.1% hyaluronidase (Sigma, H3506), and 0·02% DNAse (Roche, 04716728001)). The digestion mixture was incubated overnight at 37°C with rotation. To isolate cells from the sample, the sample was washed with primary HNC media (DMEM with 10% FBS, 1% penicillin-streptomycin, 1 µg/mL hydrocortisone, 10 µg/mL bovine insulin, and 50 ng/mL EGF). Then, the digested sample was filtered using a 40 µm cap filter and centrifuged at 400 g for 10 min. The cell pellet was washed twice with PBS and then cultured in primary HNC media in a T25 cm² flask. Cells were expanded into a new T75 cm² flask (Corning, CLS430641U), depending on confluency after 7-10 days. Fibroblasts were then isolated by subsequent quick trypsinizations (0·25% trypsin) at room temperature using cold trypsin, where fibroblasts detach faster than epithelial cells [29] (link). The recovered fibroblasts were then cultured and expanded in a T75 cm² using fibroblast media (described in the cell culture section).